The Role And Mechanism Of Neuroimmunomodulation Mediated By Spinal NF-κB/p65in The Pathogenesis Of Rat Adjuvant-Induced Arthritis

BackgroundRheumatoid arthritis (RA) is a chronic autoimmune disease that results in a chronic, systemic inflammatory disorder that may affect many tissues and organs, but principally attacks flexible (synovial) joints. It can be a disabling and painful condition, which can lead to substantial loss of functioning and mobility. The disease greatly affects the quality of life of patients. There are many drugs in the RA treatment, such as non-steroidal anti-inflammatory drugs (NSAIDs), slow-acting anti-rheumatic drugs, adrenal corticosteroids, and specific agents come into sight just these years, which cover a large part of recent research. These agents include:tumor necrosis factor (TNF)-a blocking agents, inflammatory factors specific neutralizing antibodies (interleukin (IL)-1, IL-6). A lot of effort is made to explore the pathogenesis of rheumatoid and develop new agents.It is known that peripheral tissue damage or inflammation may induce a series of activation events in the spinal cord. During the development of experimental arthritis, the peripheral nociceptors may be sensitized by inflamed synovium and damaged articular tissue, and continuous and intense nociceptive input from inflamed joints may induce neural-immune interactions. This leads to the production and secretion of cytokines, excitatory amino acids, cyclooxygenase (COX-2), and prostaglandins, which can increase the excitability of nociceptive neurons at the spinal level, including the central terminals of the primary sensory afferents (i.e., central sensitization).In addition, the spinal cord can also signal to the periphery to regulate inflammation. There is convincing evidence that a variety of spinally administered compounds attenuate peripheral inflammation. These compounds include adenosine, serotonin, ketamine and morphine, thalidomide, antagonists of microglia and inhibitors of astrocytes, and blockers of p38mitogen-activated protein kinase (MAPK). Interestingly, all these compounds have another effect in common:a reduction of neuronal excitability in the spinal cord. This common mechanism for both phenomena, i.e. for pain and maintenance of inflammation, might in fact be a clue as to the pathways involved. A variety of neuronal pathways that modulate peripheral inflammation have been implicated, including the sympathetic and the parasympathetic branches of the autonomic system. The branches of the vagal nerve and sympathetic fibers innervate immune organs, wherein they can influence peripheral immune responses. Another mechanism, the dorsal root reflex, involves antidromic signaling along somatic afferent fibers that influences peripheral inflammation by releasing neuropeptides, such as vasoactive intestinal peptide, substance P, and calcitonin gene-related peptide, from the sensory nerve endings. Moreover, the Hypothalamic-Pituitary-Adrenal (HPA) axis, which can also provide a feedback mechanism on inflammation, is often blunted in a wide range of autoimmune and inflammatory diseases such as rheumatoid arthritis.Nuclear factor-kappa B (NF-κB) is a transcription factor that plays a pivotal role in the central nervous system (CNS), in processes such as inflammation, neuronal plasticity, synaptic transmission, learning, memory, and pain(1). NF-κB contributes to the regulation of these CNS processes by positively regulating the transcription of numerous genes including cytokines (IL-1β, IL-6, and TNF-a), pro-inflammatory enzymes COX-2and inducible nitric oxide synthase (iNOS)), chemokines, and adhesion factors. Five subunits of NF-κB have been identified, namely, gp105/p50(NF-κB1), p100/p52(NF-κB2), p65(RelA), RelB, and c-Rel. The active form of NF-κB is a dimer formed from2of these subunits. The most common and best characterized form of NF-κB is the p50/p65heterodimer, which is widely expressed in the CNS and plays an important role in the regulation of gene expression. Previous studies have indicated that activation of spinal NF-KB/p65occurs in peripheral tissue damage or inflammation. In the rat chronic constriction injury (CCI) model, we previously observed extensive co-localization of NF-KB/p65with TNF-a in the spinal dorsal horn, and down-regulation of spinal NF-KB/p65expression significantly attenuated sciatic nerve ligation-induced mechanical and thermal hyperalgesia. These findings suggest NF-KB/p65has a role in central sensitization. However, it is still unknown if spinal NF-KB/p65can also facilitate a peripheral inflammatory response.In the present study, we constructed lentiviral vectors encoding short-hairpin RNAs (shRNAs) targeting NF-KB/p65(LV-shNF-κB/p65) and transfect it into rat spinal neurons and astrocytes to silence NF-KB/p65gene and to observe its inhibitory effect on the expression of IL-1β, TNF-α, and COX-2. Moreover, arthritis was induced by CFA inoculation of the rats. Rats with adjuvant-induced arthritis (AIA) were administered spinally with LV-shNF-κB/p65. Thereafter, we explored the roles of spinal NF-KB/p65in the peripheral inflammation and hyperalgesia in AIA rats. In addition, we also evaluated the expression of IL-1β,TNF-α, and COX-2in the spinal cord of these rats to gain insight into the mechanism of how NF-κB/p65contributes to peripheral inflammation and hyperalgesia in this model system. Part I Inhibitory effects of Lentivirus-mediated RNA Interference on NF-KB/p65gene in cultured Rat Spinal Neurons and AstrocytesObjectiveTo construct lentiviral vectors encoding short-hairpin RNAs (shRNAs) targeting NF-KB/p65(LV-shNF-KB/p65)and transfect it into rat spinal neurons and astrocytes to silence NF-κB/p65gene and to observe its inhibitory effect on the expression of IL-1β, TNF-a, and COX-2.Methods1. Three siRNA sequences and a negative control sequence were designed according to NF-KB/p65gene sequence of rats. The complementary DNA containing both sense and antisense DNA oligos of the targeting sequence was synthesized and cloned into the pFU-GW vector, which were digested by BamW I and EcoR I. The recombined vector was confirmed by PCR and DNA sequencing and cotransfected with pHelper1.0and pHelper2.0packaging plasmids into293T cells by use of Lipofectamine2000, then the virus titer was measured.2. Rat spinal neurons and astrocytes were cultured in vitro. Immunofluorescence staining was used to identify spinal neurons and astrocytes purity3. The recombinant lentivirus was transfected into spinal neurons and astrocytes. The transfection efficiencies were observed in each group. Thereafter, the cells were treated with1μg/ml of LPS for24h. These cells were divided into five groups:negative control group, LPS group, LPS+LV-NC group, LPS+LV-shNF-κB/p65-1group, LPS+LV-shNF-KB/p65-2group and LPS+LV-shNF-κB/p65-3group.4. Morphologic change of neurons and astrocytes were observed under invert microscop.5days after transfected, the expression level of NF-κB/p65mRNA and protein in cultured neurons and astrocytes was detected by Real-time PCR and Western Blot. ELISA was used to detect the changes of inflammatory cytokines (IL-1β,TNF-α) in the cultured astrocytes. Western blotting was used to determine the expression of COX-2in cultured neurons and astrocytes.Results 1. DNA sequencing results demonstrated that the inserted sequences were correct. The titer of virus was108-9TU/mL.2. Neurons were identitied with NSE (neuron-specific enolase) antibody. The NSE-positive cells of these cells were above85%. Astrocytes were identitied with GFAP (glial fibrillary acidic protein) antibody. The GFAP-positive cells of these cells were over90%.3. Recombinant lentivirus could be successfully transfected into spinal neurons and astrocytes with the transduction rate higher than90%. LV-shNF-KB/p65-1or LV-shNF-KB/p65-2groups showed a lower expression level of NF-κB/p65mRNA and protein in the cultured neurons (p<0.01), while LV-shNF-κB/p65-3group didn’t reach statistical significance compared with LV-NC groups (p>0.05). The interference efficiency of mRNA and protein in neurons were above95%and85%respectively. Compared to the negative control group, neuronal COX-2expression in LPS group has no significant changes (p>0.05). LV-shNF-KB/p65-1and LV-shNF-κB/p65-2can inhibit the expression of COX-2(p<0.01), but not LV-shNF-κB/p65-3(p>0.05). Compared with the LV-NC group, LPS+LV-shNF-KB/p65-1group and LPS+LV-shNF-κB/p65-2group showed a lower expression level of NF-κB/p65mRNA and protein in the cultured astrocytes (p<0.01), whereas LPS+LV-shNF-κB/p65-3group has no significant changes (p>0.05). The interference efficiency of mRNA and protein in astrocytes were above92%and83%respectively (p<0.01). Compared to the negative control group, astrocytic TNF-a, IL-1β and COX-2in LPS protein group expression was significantly increased (TNF-α,p<0.01; IL-1β,p<0.01; COX-2, p<0.05), LV-shNF-κB/p65-1and LV-shNF-κB/p65-2can inhibit this effect (p<0.01), but not LV-shNF-κB/p65-3(p>0.05).ConclusionThe lentivirus RNAi vector targeting rat spinal NF-κB/p65gene has been constructed successfully. It may down-regulate NF-KB/p65expression in spinal neurons and astrocytes and subsequent inhibiting LPS-induced spinal TNF-a, IL-1β and COX-2expression. Taken together, our reults suggest that the lentiviral vector derived shRNA approach shows a great promise for the study of functional NF-κB/p65gene expression. Part II Activation of Spinal NF-KB/p65Contributes to Peripheral Inflammation and Hyperalgesia in Rat Adjuvant-Induced ArthritisObjectiveIt is known that noxious stimuli from inflamed tissues may increase the excitability of spinal dorsal horn neurons (central sensitization), which can signal back and contribute to peripheral inflammation. However, the underlying mechanisms have yet to be fully defined. A number of recent studies indicate that spinal nuclear factor-kappaB (NF-κB) p65is involved in central sensitization as well as pain-related behavior. Thus, our aim was to determine whether NF-κB/p65also can facilitate a peripheral inflammatory response in rat adjuvant-induced arthritis (AIA).Methods1. Arthritis was induced by CFA inoculation of the rats on day0. The animals which received intrathecal10ul of the lentiviral construct encoding either the scrambled shRNA sequence (LV-NC) or encoding the NF-κB/p65shRNA sequence (LV-shNF-κB/p65) were separated into2treatment groups. One group was treated on day3, and one group was treated on day10. Thus, Rats were randomly assigned to8groups, consisting of control group, CFA group, CFA+LV-NC (3d) group, CFA+LV-shNF-κB/p65-1(3d) group, and CFA+LV-shNF-κB/p65-2(3d) group, CFA+LV-NC (10d) group, CFA+LV-shNF-KB/p65-1(10d) group, and CFA+LV-shNF-KB/p65-2(10d) group.2. In order to confirm the knockdown of NF-κB/p65expression by spinally delivered LV-shNF-κB/p65, on day14, the expression and activation of spinal NF-κB/p65was assayed by Western blot analysis, immunofluorescence, and electrophoretic mobility shift assay (EMSA). Pain-related behavior and paw swelling were assessed at baseline (1day before CFA injection) and day0was the time point of CFA injection. Measurements were also obtained at6,8,10,12,14,16,18and20days following CFA injection. On the day20, joint histopathological changes were evaluated. Moreover, the expression of spinal TNF-a, IL-1β and COX-2were assessed at14days after CFA treatment.Results 1. Peripheral Inflammation induced an increase in NF-KB/p65expression in the spinal cord, mainly in the dorsal horn neurons and astrocytes. Spinally delivered LV-shNF-KB/p65-1and LV-shNF-κB/p65-2both knocked down NF-κB/p65expression (p<0.01).2. CFA group showed an obvious increase in mechanical hyperalgesia, thermal hyperalgesia and paw edema compare to control group. The CFA+LV-shNF-κB/p65-1and CFA+LV-shNF-KB/p65-2groups treated on day3or10can inhibite this effect(all p <0.01). The severity of Joint inflammation and destruction was significantly lower in the group treated with LV-shNF-κB/p65-2on day3, whereas these changes were modestly, but not significantly, less severe in the group treated with LV-shNF-KB/p65-1on day3(p>0.05). Furthermore, the histologic features of arthritis were not statistically significantly different between the CFA+LV-NC (10d) group and the CFA+LV-shNF-κB/p65-1(10d) or CFA+LV-shNF-KB/p65-2(10d) groups (p>0.05). The IL-1β and TNF-a protein levels in the CFA+LV-shNF-KB/p65-1(3d) and CFA+LV-shNF-κB/p65-2(3d) groups were significantly lower than those in the CFA+LV-NC (3d) group (p<0.01). Moreover, TNF-a expression levels in the CFA+LV-shNF-KB/p65-1(10d) and CFA+LV-shNF-κB/p65-2(10d) groups were also obviously lower than those in the CFA+LV-NC (10d) group (p <0.05). Furthermore, when compared with the CFA+LV-NC group (10d), the expression of IL-1β was observed to be significantly lower in the CFA+LV-shNF-κB/p65-2group (10d)(p<0.05), whereas the expression of IL-1β was modestly, but not significantly, lower in the LV-shNF-κB/p65-1(10d) group (p>0.05). Moreover, the LV-shNF-κB/p65-2group showed a decreased expression of COX-2protein (p<0.05), while the LV-shNF-KB/p65-1group (3d) showed a modestly reduced expression of COX-2protein, but the difference did not reach statistical significance (p>0.05). In addition, in the CFA+LV-shNF-KB/p65-1and CFA+LV-shNF-κB/p65-2groups treated on day10, the COX-2protein levels were also not statistically significantly different when compared with those in the CFA+LV-NC group (10d)(p>0.05).ConclusionPeripheral Inflammation induced an increase in NF-KB/p65expression in the spinal cord, mainly in the dorsal horn neurons and astrocytes. Spinally delivered LV-shNF-KB/p65knocked down NF-κB/p65expression and significantly attenuated hyperalgesia, paw edema, and joint destruction. In addition, spinal delivery of LV-shNF-κB/p65reduced the over-expression of spinal TNF-α, IL-1β and COX-2. These indicate that spinal NF- KB/p65plays an important role in the initiation and development of both peripheral inflammation and hyperalgesia. Thus, inhibition of spinal NF-κB/p65expression may provide a potential treatment to manage painful inflammatory disorders.